Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA

Abstract

PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)--a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub-PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provide unexpected insight into previously unexplained biological observations.

Fig. 1.

SAXS analysis of PCNA–Ub in solution suggests that ubiquitin is not exclusively oriented in the position determined by crystallography. (A) SAXS curves of split-fusion and cross-linked PCNA–Ub overlaid on the calculated curve for PCNA–Ub trimer from the crystal structure 3L10.pdb. (B) Guinier analyses of SAXS data showing relative linearity of sample in Guinier region, indicating lack of aggregation in sample. (C) Ab initio shape predictions calculated from experimental scattering curves of split-fusion or cross-linked PCNA–Ub suggests only one or two ubiquitin positions are extending away from the PCNA ring.

Fig. 2.

Tethered Brownian dynamics simulation shows the range of covalently bound Ub positions on the surface of PCNA. The positions of Ub heavy atoms (C,N,S, and O) in 6,837 frames from a 34-μs TBD simulation were binned and displayed relative to PCNA as a 3D histogram.

Fig. 3.

Three positions of ubiquitin derived from multiscale refinement. The three computationally derived positions (blue, orange, red) are shown relative to PCNA (gray) and to the ubiquitin position determined in the crystallographic studies (black). The computationally derived positions cover up biologically important positions (G178 and E113) identified in yeast mutation studies and the J and P loops that are thought to be important structurally for ubiquitin and sumo positioning.

Fig. 4.

An MES ensemble of both discrete and flexible positions of ubiquitin relative to PCNA best fit the experimental SAXS data for split-fusion (green) and cross-linked (blue) PCNA–Ub. (A) Schematic showing MES methodology. One hundred thirty PDB models were generated where the three ubiquitins per PCNA homotrimer were placed at the crystallographic (x) position, the MD-identified positions (a,b,c), or the BILBOMD-generated flexible (f). Ensembles of three models were then compared to the experimental SAXS data in FOXS. (B) The scattering curve of the best MES ensemble fits the experimental scattering data better than the crystal structure 3L10.pdb. (C) P(r) plots showing the good fit of the MES ensemble to the experimental data. (D) The three models that as an ensemble best fit the experimental scattering curve are shown in ribbon models. Relative proportion of each position in x, a/b/c, or f shows that ubiquitin adopts both the crystallographic and computationally determined discrete positions as well as being flexible in solution.

Fig. 5.

Possible biological significance of the different ubiquitin positions relative to PCNA. The observation that covalently attached ubiquitin can dynamically occupy different positions on PCNA allows a tool belt model for TLS polymerase binding and functions.